Neurosteroids are reduced in diabetic neuropathy and may be associated with the development of neuropathic pain
Author(s)
Humble, Stephen R
Type
Journal Article
Abstract
Introduction: Peripheral and central sensitisation are implicated in the development of neuropathic pain. Hypersensitivity of pain pathway neurons has been described in animal models of diabetic neuropathy, which is postulated to be related to an imbalance between inhibitory and excitatory signals within the spinal cord. GABAergic neurons within the pain pathway are vital for the transmission of painful stimuli to higher centres. A developmental change in the rate of exponential decay of GABAergic synaptic events has been observed in other types of neurons and this may be associated with fluctuations in endogenous neurosteroid tone.
Methods: The whole-cell patch-clamp technique was used on slices of neural tissue. Electrophysiological recordings were obtained from wild type mice between the ages of 6 and 80 days in the spinal cord, the nucleus reticularis of the thalamus and the cerebral cortex. Recordings were also obtained from mice with diabetic neuropathy (ob/ob and db/db) between the ages of 60 and 80 days. Behavioural experiments were performed to examine mechanical and thermal nociception.
Results: Electrophysiological recordings from cortical pain pathway neurons from mature type-2 diabetic mice revealed that the endogenous neurosteroid tone is reduced compared to control. However, selected neurosteroid compounds had a more pronounced effect on the GABAA receptors of these diabetic mice. ob/ob mice exhibit mechanical hyperalgesia and allodynia, which was reduced by neurosteroids applied exogenously.
Conclusions: The reduced endogenous neurosteroid tone in ob/ob mice may be linked to their hypersensitivity. Neurosteroids may exert analgesic effects in pathological pain states by attempting to restore the physiological GABAergic inhibitory tone.
Methods: The whole-cell patch-clamp technique was used on slices of neural tissue. Electrophysiological recordings were obtained from wild type mice between the ages of 6 and 80 days in the spinal cord, the nucleus reticularis of the thalamus and the cerebral cortex. Recordings were also obtained from mice with diabetic neuropathy (ob/ob and db/db) between the ages of 60 and 80 days. Behavioural experiments were performed to examine mechanical and thermal nociception.
Results: Electrophysiological recordings from cortical pain pathway neurons from mature type-2 diabetic mice revealed that the endogenous neurosteroid tone is reduced compared to control. However, selected neurosteroid compounds had a more pronounced effect on the GABAA receptors of these diabetic mice. ob/ob mice exhibit mechanical hyperalgesia and allodynia, which was reduced by neurosteroids applied exogenously.
Conclusions: The reduced endogenous neurosteroid tone in ob/ob mice may be linked to their hypersensitivity. Neurosteroids may exert analgesic effects in pathological pain states by attempting to restore the physiological GABAergic inhibitory tone.
Date Issued
2016-08-05
Date Acceptance
2016-08-05
Citation
F1000Research, 2016, 5
ISSN
2046-1402
Publisher
F1000 Research Ltd
Journal / Book Title
F1000Research
Volume
5
Copyright Statement
Copyright: © 2016 Humble SR. This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
License URL
Sponsor
GlaxoSmithKline Services Unlimited
Grant Number
COL011953
Notes
referee-status: Indexed referee-response-15694: 10.5256/f1000research.9720.r15694, Christopher Connolly, Centre for Environmental Change and Human Resilience (CECHR), University of Dundee, Dundee, UK, 15 Aug 2016, version 1, indexed referee-comment-2500: <b>Stephen Humble</b>; <i>Posted: 17 Feb 2017</i>; Authors Comments in response to the reviewer Thank you very much indeed for talking the time to review the paper 1. The author agrees that development does not fluctuate. Neurosteroid tone starts out very high in the most immature neurons then decreases as with maturity. At around P20 the neurosteroid tone is almost negligible, but a modest neurosteroid tone has reappeared in mature neurones by P60 (Humble 2013, Brown 2012). The presence of the neurosteroid tone may be revealed with the use of cyclodextrin. Separately the overall τ <sub>W </sub>of the mIPCs also decreases independently of the neurosteroid tone with progressive maturity (Humble 2013, Brown 2012). 2. Loss of neurosteroid tone in development thought to be due to reduced production. But separately there is also changes in the expression of different receptor subtypes (e.g. alpha-2 as per Bosman <i>et al.,</i> 2005). 3. I can insert: ‘The use of small cyclodextrins was used to attempt to control for the generic effects of cyclodextrin such as an interaction with the cholesterol within the neuronal membrane.’ 4.The author had not performed experiments using isolated neurons in culture, nor explored specific potential changes in trafficking/gene expression. The lack of these experiments could be considered a relative limitation of the study and would be a useful complimentary study. The author used different methods of neurosteroid presentation in order to explore the issue of diffusion and ensure that neurosteroid penetration of the slice was optimal in each situation. In order to maximize the neurosteroid effect the slices were incubated more than an hour with the relevant neurosteroid compound as per work on other lipophilic GABAergic compounds by other authors (Benkwitz <i>et al.,</i> 2007; Gredell <i>et al.,</i> 2004) 5. The lack of effect of finasteride on WT and ob/ob mice is not sufficient to make the statement that there is no endogenous basal neurosteroid-induced tone. Indeed, there is in fact an endogenous basal neurosteroid-induced tone as illustrated by the experiments with cyclodextrin. The lack of observed effect with finasteride is consistent with other authors (Brown, 2012 Thesis). The lack of effect with finasteride in this context is explained as follows: Finasteride inhibits the production of new neurosteroid via 5α-reductase inhibition. However it does not impact on the endogenous neurosteroid compounds that are already present within the slice preparation. This question was considered during the project and it was decided that the appropriate way to answer this question would be to inject finasteride in vivo, some hours/days prior to the in vitro experiments. The half-life of the neurosteroid Allopregnanolone is approximately 30 minutes (Mellon et al., 2008). However, a functional equilibrium typically exists between Allopregnanlone and its neurosteroid precursors which act as an immediate reservoir that would not be blocked instantly by finasteride, which inhibits 5αR rather than 3α-HSD (Figure 1). 6. It is very interesting 7. Agreed. The rotarod test results for the ob/ob are included for completeness and for demonstrating the phenotype but have minimal validity other than that. The ob/ob rotarod experiments illustrate why the sedative effects of neurosteroid were studied in WT rather than the ob/ob. The ob/ob is type-2 diabetic because of obesity. Other obese mouse models such as the db/db also have type-2 diabetes. There does not appear to be an obese mouse model without type-2 diabetes. Thus the respective impacts of obesity and type-2 diabetes on rotarod ability cannot be differentiated. 8. This is a potentially translatable finding. 9. It is known that general affinity for the GABAA receptor varies between different neurosteroid compounds. Separately, GABAA receptors expressing specific subunits have greater affinity for a given neurosteroid. Indeed, the following is an extract from my thesis: ‘THIP is a low affinity partial agonist at receptors expressing the g subunit ( <i>i.e.</i> most GABA <sub>A</sub>Rs), but has high affinity for GABA <sub>A</sub>Rs that express the a4b3d subunits where it behaves as a ‘superagonist’ compared to GABA (Brown <i>et al.,</i> 2002; Farrant & Nusser, 2005; Krogsgaard-Larsen <i>et al.,</i> 2004). d-GABA <sub>A</sub>Rs are predominantly located at extrasynaptic locations; therefore THIP is a useful pharmacological agent for the selective activation of tonic currents (Belelli <i>et al.,</i> 2005; Farrant & Nusser, 2005).’ (Humble, 2013) For reasons of brevity the issue of differential binding sites to neurosteroids was not explored significantly within the paper, but this was covered in substantial detail within my thesis (Humble, 2013). It is summarised in the Conclusions section of the thesis: ‘Pipette-applied neurosteroids allopregnanolone and ganaxolone (but not DHP) were also able to prolong the decay time of cortical GABA <sub>A</sub>R mIPSCs in a differential manner. Allopregnanolone induced a greater effect than ganaxolone on GABA <sub>A</sub>R mIPSC decay time, but there was no difference in the response to the respective drugs between the WT and <i>ob/ob</i> mice. These findings suggest that neurosteroids are able to modulate the GABA <sub>A</sub>R from the intracellular compartment and that the sensitivity of the GABA <sub>A</sub>R is the same for WT and <i>ob/ob</i> mice. In addition, when indometacin was co-applied in the pipette with ganaxolone it had no impact on the efficacy of ganaxolone. This observation is inconsistent with the notion that indometacin competes with ganaxolone for its binding of the GABA <sub>A</sub>R.’ (Humble, 2013) 10. This is an excellent point and is the subject of a Brief Communication that I have written on the subject of Mitochondrial dysfunction and neurosteroid synthesis in diabetic neuropathy that will be published shortly. I would be happy to refer to this further in the discussion. referee-response-19599: 10.5256/f1000research.9720.r19599, Anthony Dickenson, Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK, 14 Feb 2017, version 1, indexed referee-comment-2576: <b>Stephen Humble</b>; <i>Posted: 22 Mar 2017</i>; Dear Reviewer, thank you for all your comments! referee-response-20664: 10.5256/f1000research.9720.r20664, Xue-Jun Song, Changyu Jiang, Center for Anesthesiology and Pain Medicine, Department of Anesthesiology, Peking University Cancer Hospital and Institute, Beijing , China, 03 Mar 2017, version 1, indexed referee-comment-2577: <b>Stephen Humble</b>; <i>Posted: 22 Mar 2017</i>; 1. Dear Reviewer, thank you for all your comments. This is an excellent point. The next logical step would be to carry out mass spectrometry to compare ob/ob and WT samples from pain pathway tissue. Unfortunately this was beyond the scope of the project. 2. GABA A receptors are the principle targets for the neurosteroid molecules that were investigated in these studies. The difference between ob/ob, db/db and WT mice is as follows: ob/ob lack leptin, db/db lack a functional leptin receptor and WT have normal leptin secretion and normal leptin receptor. The leptin receptor is entirely distinct from the GABA A receptor and there are no credible reports of leptin having a significant impact on the GABA A receptor. In anticipation of this question the second type 2 diabetic model (db/db) was used in order to illustrate that leptin was not implicated in the results. There is no specific reason a priori to consider that the GABA A receptor structure and function would be congenitally different than the WT. Indeed, the diabetic neuropathy happens with advancing maturity- hence why the experiments were carried out in mature animals that had developed neuropathic changes. The experiments using intracellular cyclodextrin illustrate that when the entire neurosteroid tone is removed by sequestration the baseline GABA A receptor function is the same for all strains of mice including diabetic and WT. Secondly, the application of the active neurosteroid allopregnanolone had the same impact on all strains. Thirdly, the intracellular application of neurosteroids enhanced GABA A receptor function by a similar margin in WT and ob/ob. All these observations taken together appear to be inconsistent with the hypothesis that GABA A receptor sensitivity differs between ob/ob, db/db and WT. If the GABA A receptor sensitivity is unchanged between strains this would be consistent with the hypothesis that GABA A receptor expression was not significantly different in the ob/ob, db/db and WT. 3. Due to the large amount of data contained within these studies the tables containing information such as the frequency of the mIPSCs are included as supplementary files. They can be accessed on the article’s webpage immediately below the Supplementary Material section and immediately above the References section. Any additional material not found there will be found in the author’s thesis using the link below: http://discovery.dundee.ac.uk/portal/en/theses/neurosteroids(c4659466-cd41-494d-aec6-edcf50e5274b).html In general terms, the frequency of mIPSCs increased with development in pain pathway neurons. This was anticipated due to known progressive synaptic development with maturation and was consistent with work from other authors. Specifically, there were no unexpected findings related to frequency with development. In addition, the neurosteroids investigated had no significant, consistent impact on frequency in the neurons studied. For these reasons, frequency has not been discussed in depth in the paper despite the fact that the frequency of the mIPSCs was recorded and analyzed routinely as part of the experiments along with other parameters. 4. This is an interesting observation. It is most likely due to differences in the utilization of the electrophysiological equipment and the process of analyzing the mIPSCs. It is not uncommon to observe differences between institutions for these reasons and there are many examples within the international literature. It is worth noting that the parameters such as amplitude, frequency, charge transfer, decay time in control recordings was consistent with recordings made by colleagues within the same institution that were using the same equipment set up and analysis protocols and software. In addition, it is standard established practice within the institution to exclude mIPSCs with a Rise Time greater than 1 millisecond (as mentioned in the Data Analysis section of the Methods). This approach excludes events (which have a longer Rise Time) because they are not immediately adjacent to the synapse. The author notes that the excluded events typically have lower amplitudes and have a morphology less like typical mIPSCs. 5. I have checked and rechecked the alphabetical sequences of the figures and figure legends and unfortunately cannot detect a specific issue. Please indicate which Figures and Figure Legends are the problem and I will correct them. grant-information: This research was supported by the Wellcome Trust (Grant No. 090667).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. copyright-info: This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Publication Status
Published
Article Number
1923
Date Publish Online
2016-08-05